The Human Eye
The structure of the ocular surface is described in FIG. 1. The ocular surface comprises:                cornea 109,        conjunctiva 108,        tear film 103, and        upper eyelid 101 and lower eyelid 105.        
The cornea 109 is a transparent tissue which role is to catch and focus light on the eye transparent crystalline structure or lens.
The conjunctiva 108 is the outermost layer of the eye 111 and the inner surface of the eyelids 101 and 105. Conjunctiva 108 is covering the white part of the eye 111 or sclera. The limbus 113 is the border of the cornea 109 and the sclera.
Tear film 103 is composed of 2 layers: outermost lipid layer derived from the meibomian glands and innermost aqueous layer composed of lacrimal fluid mixed with soluble mucins. Eyelids 101 and 105 include the lachrymal glands secreting the aqueous component of the aqueous layer (mucins being produced by the goblet cells of the conjunctiva), and the meibomian glands secreting the lipid layer.
Upper eyelid 101 and lower eyelid 105 are the first line of defence of the visible ocular surface, followed by tear film 103. On the last defence lines are the two distinct tissues conjunctiva 108 and cornea 109. The ocular surface is a mucosal transition between the external environment and the fragile intraocular structures. Any condition that reduces the production, alter the composition, or impede the distribution of the tear film 103 may cause noticeable irritations to the structures of the front surface of the eye 111 and a degradation of vision.
These conditions are often related to problems with the structure or function of the eyelids 101 and 105, the conjunctiva 108, or the cornea 109. If not timely and properly diagnosed, and depending upon the severity of symptoms, individuals may be at increased risk of developing secondary infection or chronic inflammation that may not respond to treatment.
Most of the tear fluid is stored in the conjunctival sac 107, which is the transition portion which forms the junction between the posterior surface of the lower eyelid 105 and the surface of the eye 111. The surface of the conjunctival sac 107 is covered by the conjunctiva 108. The bottom of the conjunctival sac 107 is called conjunctival cul-de-sac.
Due to anatomical constraint, a maximum of 30 μl of tear fluid is estimated to be possibly held by the human eye without overflow or spillage. The normal resident tear volume in the eye is approximately 7.5 μl, most of which resides in the conjunctival sacs with approximately 1 μl covering the cornea.
Tear turnover rate in human is about 0.16/min, that is to say that each minute, 16 percent of the tear volume is renewed. It corresponds to around 1.2 μl per minute.
As mentioned before, the structure and composition of the tear film 103 can be divided into two main layers.
The innermost layer in contact with the conjunctiva 108 is the aqueous layer. The aqueous layer is a mixture of lacrimal fluid and different soluble mucins, which are glycosylated proteins.
The aqueous layer is important in that it provides a protective layer and lubrication to prevent dryness of the eye. Dryness of the eye can cause symptoms such as itchiness, burning, and irritation, which can result in discomfort. It is a few micrometers thick.
The outermost lipid layer is comprised of many lipids known as meibum or sebum. This outermost lipid layer is very thin, typically less than 250 nm thick. The lipid layer provides a protective coating over the aqueous layer to limit the rate at which the underlying aqueous layer evaporates. A higher rate of evaporation of the aqueous layer can cause dryness of the eye. The lipid layer also lubricates the eyelids 101 and 105 during blinking, which prevents dry eye.
More generally, in the human eye, the unique structure of the tear film 103 enables it to perform many functions, based on its lipid, and aqueous layer components.
Seven major functions of the tear film 103 are:                Maintaining a smooth surface for light refraction. The tears form the first refractive surface encountered by light on its path to the retina. For clear vision, it is critical to maintain the transparency of the second refractive surface that rays of light encounter, i.e. the cornea 109.        Lubricating the eyelids 101 and 105.        Lubricating the conjunctiva 108 and the cornea 109, thus avoiding ocular surface mechanical damage from the high pressures generated by each blink.        Supplying the cornea 109 with nutrients by transporting oxygen and a limited number of other nutrients to the avascular cornea 109, regulating the electrolyte composition and pH.        Providing white blood cells with access to the cornea 109 and conjunctiva 108.        Removing foreign materials from the cornea 109 and conjunctiva 108. The tear film 103 protects the ocular surface from the external environment by responding dynamically to a wide range of external conditions and potentially damaging situations. These external stresses include desiccation, bright light, cold, mechanical stimulation, physical injury, noxious chemicals, and bacterial, viral, and parasitic infection.        Defending the ocular surface from the pathogens via specific and nonspecific antibacterial substances.Example of Ocular Surface Diseases and DiagnosisDry Eye Disease        
Dry eye disease, or keratoconjunctivitis sicca (KCS), is a common eye problem that involves irritation and blurry vision caused by damage to the ocular surface by insufficient tear production or excessive tear evaporation and that can affect quality of life. In KCS, one or more of the tear film 103 structure components is present in insufficient volume or is otherwise out of balance with the other components.
It is prevalent in the ageing population, with a higher incidence among women. Although it is a common diagnosis, physicians have to rely on many symptoms and diagnostic tests to confirm its presence.
Schirmer Test and Dry Eye Diseases
The Schirmer test estimates the tear volume or secretion. It involves a strip of filter paper partially blocked in the lower conjunctival sac 107, inducing irritation and reflex tearing. The Schirmer test relies on the presence of a good tear meniscus to act as a reservoir from which fluid can be drawn and absorbed by the paper.
Unfortunately, the Schirmer test has many disadvantages, including low reproducibility, sensitivity and specificity, frequent discomfort, difficulty of performing the test in children, potential injury of the conjunctiva 108 and cornea 109, lack of a definite site of paper placement in the conjunctival sac 107, uneven absorption of tear fluid by the paper strip, uncertainty whether the quantity of fluid absorbed by paper strips is directly proportional to the wetted length, difficulty for evaluating the wetting length in cases where the leading edge of the wetted area is round or oblique, and lack of control over reflex lacrimation.
Osmolarity and Dry Eye Diseases
It is known that the fluid tonicity or osmolarity of tears generally increases in patients with KCS. KCS is associated with conditions that affect the general health of the body, such as Sjogren's syndrome, ageing, and androgen deficiency. Therefore, tear film 103 osmolarity can be a sensitive and specific indicator for the diagnosis of KCS and other conditions.
The osmolarity of a sample tear fluid can be assessed by an ex vivo technique called “freezing point depression”, taking advantage of the fact that solutes or ions in a solvent cause a lowering of the fluid freezing point from what it would be without the ions. Presently, freezing point depression measurements are made ex vivo by removing tear samples from the eye 111 using a micropipette or a blunt needle and measuring the depression of the freezing point of the sample that results from heightened osmolarity.
However, these ex vivo measurements are plagued by many difficulties. For consistent analysis, a relatively large volume should be collected, at least around 20 μl of tear sample, typically around 60-80 μl. Moreover, corresponding processes according to the state of the art stated above require the presence of medical personnel for about one hour. Tear turnover is approximately 42 percent lower in KCS subject than in normal subject. Hence the need for at least twice the time to collect the necessary amount from KCS subject than for normal subject. Because no more than about 10 to 100 nL of tear sample can be obtained at any one time from a KCS subject, the collection of sufficient amounts often requires a physician to induce reflex tearing in the subject. Reflex tears are more dilute, i.e. have fewer solute ions than normal tear fluid. In some cases, reflex tearing produce false negatives.
False negatives may also result from the inherent metastability of the tear film 103 of KCS subject. This metastability, punctuated by periods of hyperosmolarity may be explained by the subject compensatory mechanisms, such as increased blinking and reflex stimulations of the eye glands, punctually subdued by breakdowns. Any dilution of the tear film 103 or reunion of samples taken at different times invalidates the diagnosis of an osmolarity test for KCS and therefore makes currently available ex vivo methods prohibitive in a clinical setting.
The absolute validity of an osmolarity based KCS diagnosis is hence in itself to be questioned as the statistic osmolarity profiles of KCS subjects and normal subjects overlap each other, resulting in a zone of non-robust diagnosis, as described in “Tear Film Osmolarity: Determination of a Referent for Dry Eye Diagnosis” by Tomlinson et al. in “Investigative Ophthalmology a Visual Science” 47 (2006) 4309-4315.
Allergy
Allergic diseases of the conjunctiva are a common and heterogenous group disorders characterized by the expression of a classical type 1 IgE-mediated hypersensitivity reaction at the conjunctival level.
IgE is a subclass of immunoglobulin and is involved in the pathophysiology of severe type 1 hypersensitivity allergic reaction. IgE is detected in circulatory blood and somatic tissues. IgE in tissues binds to the IgE receptor expressed mainly on mast cells. Clinical examinations to detect IgE are classified into two methods: determination of total IgE and determination of antigen-specific IgE in serum. Total IgE in serum reflects systemic atopic diathesis and antigen-specific IgE indicates the presence of a sensitized antigen.
As described in “Clinical Evaluation of Total IgE in Tears of Patients with Allergic Conjunctivitis Disease Using a Novel Application of the Immunochromatography Method” by Inada et al. in “Allergology International” 58 (2009) 585-589, for many allergic disorders, such as allergic conjunctivitis, eye-specific immune reaction is considered to be critical in the pathogenesis.
To investigate the eye-specific immunological disorders in allergic diseases, examination of total IgE in tears might be a better marker to assess the tissue-specific pathophysiology rather than serum IgE examination. Therefore, an examination of total IgE in tears is useful not only for the diagnosis but also for the assessment of allergic conjunctivitis diseases severity.
For example, total IgE serum levels are significantly increased in vernal KCS than in controls. However, IgE levels are variable among subjects with ocular allergy and cannot be used as a reliable indicator of disease activity or severity.
Studies investigating allergen-specific serum IgE levels have detected a range of allergen specificities. Allergen specific IgE is also increased in tear samples and there is a highly significant correlation with ocular allergy symptoms, supporting a diagnostic value for specific tear IgE, although limitations in volume of above described tear sampling methods restrict its use in routine immunoassays.
Proteomics
Human tear fluid is shown to have more than 600 proteins, among which 491 proteins have been duly identified. The tear proteins play an important role in maintaining the ocular surface, and changes in tear protein composition may reflect the changes in the health of the ocular surface.
The relative proportions of the proteins present in a subject tear sample, that is to say its tear fluid proteomic profile, depend on the method of tear collection. Invasive methods, including filter paper and cellulose sponges, stimulate the conjunctiva 108, induce serum leakage, and result in a higher proportion of plasma proteins. Samples collected by less invasive means, such as fine capillary tubes dipped into the tear meniscus, demonstrate a higher proportion of lacrimal gland proteins.
As described in “Comparative proteomics of human male and female tears by two-dimensional electrophoresis” by Ananthi et al. in “Experimental Eye Research” 92 (2011) 454-463, proteomic analysis of tear fluid shows promising results for eye diseases diagnostic.
Tear fluid analysis can be based on noninvasive approaches in early diagnosis and study of pathogenesis of eye-related diseases. It may also assist follow-up assessment of therapeutic treatment. For some eye diseases such as KCS, the development of new potential treatments is hampered by the fact that there are no objective criteria available to precisely assess the treatment.
A standard tear proteomic pattern from healthy individuals may serve as a reference to measure the success of treatment. Tear proteome profiling can also generate useful information for the understanding of the interaction between an eye and its contacting objects, such as a contact lens or a lens implant. This is important for designing improved eye-care devices and maintaining the health of an eye.
As described in “Diagnostic biologique des conjonctivites” by Batellier et al. in “EMC, Ophtalmologie” 21-130-B-10 (2010), IgE and proteomic analyses may provide consistent diagnoses, provided a sufficient amount of tear fluid can be collected.
U.S. Pat. No. 7,810,380 discloses a system and method for collecting tear film 103 and measuring tear film 103 osmolarity that requires only a small amount of tear film 103, typically around 20 nL. But this method requires a system that is expensive and complicated to produce, and this system requires the presence of a physician for being used. Moreover, the validity of a diagnosis that depends on a measurement performed on such a small amount of tear fluid sampled at a precise moment is questioned. A gradient of composition exists within the few micrometers thick tear film 103, and the composition also differ within the tear meniscus. This results in different osmolarity measures depending on the exact spot, position of sampling, as well as the pressure applied and the angle of the device chosen by the person manipulating the device. The gradient is therefore questioning the reproducibility of such a test. A diagnosis resulting from such a sample hence strongly depends on the circumstances of the time of the sampling, and may not represent the eye state.
RU2335233 discloses a device for tear collection. However this device does not solve tear sampling main problems in that it just allow to collect tear fluid already evacuated from the eye and does not prevent evaporation during tear sampling that will modify the tear composition.
Thus, current sampling techniques are of questioned reproducibility or unavailable in a clinical setting and can't attain the volumes necessary for many ocular surface disorders diagnostics, typically 60-80 μl. Even if the amount was reached, the necessary time would induce evaporation effect, thus impairing the quality of the sample. There is also a risk of corneal or conjunctival injury or irritation of the conjunctiva 108 due to the repetitive use of collection devices such as capillary tubes, surgical sponges or tear strips.
Hence the need for an improved and clinically feasible sampling technique.